The goal of this project is to develop a plasmid-based therapy that will destroy the integrated HIV-1 DNA (HIV- 1 provirus) in infected cells. Current HIV-1 anti-retroviral therapy (ARV) consists of a cocktail of drugs that blocks viral binding and replication, but does not destroy the HIV-1 provirus. In addition, ARV does not impact the large reservoir of latent HIV-infected cells present in lymphoid tissues and in the gut of infected patients. Moreover, patients must remain on ARV for life, or viral replication will resume once treatment is interrupted. We postulate that cleaving one or more regions within the HIV-1 provirus will impair the ability of an infected cell to transcribe viral genes and to make new vira RNA genomes, and subsequently prevent the production of infectious virions. In addition, this approach would also target and destroy the HIV-1 provirus in latent HIV-infected cells, a population of cells for which anti-retroviral therapy is ineffective. Our approach involves adaptin an immune defense mechanism used by the majority of prokaryotes to block infection by viruses. This defense mechanism is called CRISPR/Cas, in reference to the two major components of this system. The first component is an RNA molecule termed CRISPR RNA, or crRNA, transcribed from the prokaryotic CRISPR locus, and which is complementary to a region in the invading viral genome. The second component is a nuclease enzyme termed Cas (for CRISPR-associated) that is encoded from the cas locus and which binds to the crRNA to form a hybrid molecular complex. Key to this approach is the design of specific guide RNAs that not only are complementary to a unique region in the target gene, but also do not bind to any other region in the genome. We have identified and cloned 9 different guide RNAs that meet all of the requisite criteria for binding to the HIV-1 proviral DNA sequence: they bind to a unique 20 nucleotide region in the HIV provirus, they recognize a region that meets the requirement for the Cas endonuclease activity, and they do not recognize any other region in the human genome, including other endogenous retroviral sequences. In Phase I we will: (1) modify the sequence of the plasmids that produce guide RNAs complementary to unique regions within the HIV-1 proviral DNA in order to increase expression and hybridization with the humanized Cas9 protein, and (2) determine the extent to which the guide RNA:hCas9 hybrid molecular complex binds to and degrades the HIV-1 proviral DNA in HIV-infected macrophages, a primary cell type that represents a pool of latent HIV-infected cells [6]. Our long-term objective is to develop a therapy that can be used in conjunction with existing ARVs to not only prevent the production of infectious virions from infected cells, but that would target the large reservoir of HIV-1 infected cells, and obviate the need for life-long ARV treatment.